Throughout this application, certain publications are referenced by number. Full citations for these publications may be found listed at the end of the specification and preceding the Claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art. A Sequence Listing is provided.
The present invention relates generally to to multi-drug resistance, and more particularly to materials such as multi-drug resistant proteins (MRP), and to possible diagnostic and therapeutic uses thereof. The invention further relates to methods for identifying treatments refractive to drug resistance.
Microbial and cellular resistance to drug therapy is a major and long-standing problem to the treatment of disease and infection, including cancer. Cross-resistance between different anti-microbial and anti-cancer agents, which are structurally and functionally distinct, is a relatively common phenomenon called multi-drug resistance (MDR).
With respect to cancers, some malignant tumors respond poorly to chemotherapy, indicating that the target cells are intrinsically resistant. Other tumors initially respond well to chemotherapy, but appear to develop resistance, indicating a selection process or cellular response to the chemotherapeutic agent(s). The broad-spectrum resistance characteristic of MDR, therefore, is of great clinical significance.
MDR was initially described in cultured tumor cells which following selection for resistance to a single anti-tumor agent became resistant to a range of chemically diverse anti-cancer agents (52). These MDR cells exhibited a decrease in intracellular drug accumulation due to active efflux by transporter proteins. The so-called xe2x80x9cmulti-drug transportersxe2x80x9d are membrane proteins capable of expelling a broad range of toxic molecules from the cell (53). These multi-drug transporters belong to the ATP-binding cassette (ABC) superfamily of transport proteins that utilize the energy of ATP hydrolysis for activity (53, 57). In microorganisms, multi-drug transporters play an important role in conferring antibiotic resistance on pathogens.
Several mechanisms have been described as responsible for MDR. The most well characterized gene conferring drug resistance by an ATP-dependent efflux mechanism is the MDR1 gene product, P-glycoprotein (Pgp). a member of the ABC cassette family of transporters. Pgp removes hydrophobic drugs of diverse chemical structures from cells as an efflux pump (55).
Another transporter protein, capable of conferring drug resistance, the multi-drug resistance protein (MRP), has been identified in a number of MDR human tumor cell lines that do not appear to express Pgp (52). The presence of MRP at the cell surface of such cells has been associated with alterations in drug accumulation and distribution (52). Expression of MRP causes a form of multi-drug resistance similar to that conferred by Pgp (52). The two proteins, however, are only distantly related. MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates. Like Pgp, MRP has a broad substrate specificity. In addition to hydrophobic compounds, MRP is able to transport metallic oxyanions and glutathione and other conjugates, including peptidyl leukotrienes (52). This is in contrast to Pgp. (Stride, B D, et al., 1997, Mol. Pharmacol., 52:344-53). The mechanism by which MRP transports these compounds and mediates multi-drug resistance is not understood. In addition, topoisomerase II has been associated with MDR. Like Pgp, MRP is expressed in normal human tissues in addition to tumor cells (52). In normal cells, MRP appears to be located within the cytoplasm, indicating that it may function differently in normal cells as compared with tumor cells (52). Homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, candida albicans, Saccharomyces cerevisiae and Lactococcus lactis (53).
Although MDR1 was cloned some time ago, proteins in animal cells that were functionally similar were not readily identified. MRP has been described which in some cell types confer a drug resistance phenotype similar to the MDR1 gene (58). The prototype MRP1 gene was first described in 1992. Subsequently, MRP2 (cMOAT) was cloned. Both MRP1 and MRP2 act to efflux anionic compounds, including drugs or endogenous compounds. Several yeast MRP homologues have been identified (49) and recently, additional human homologues have been identified in the EST databases. Particularly, Borst and colleagues searched the EST database and identified four additional family members (MRP2, MRP3, MRP4, and MRP5). Nonetheless, the human MRP homologues have until now remained functionally undefined. MRP3 has been described as exhibiting high expression in some cell lines but not in others, with overexpression of MRP3 in resistant lines being identified in several doxorubicin-resistant and cisplatin-resistant cell lines (49). MRP5 was identified as being very widely expressed. MRP4 in contrast to MRP3 and MRP5, was not reportedly overexpressed in any cell line analyzed (49). Importantly, the EST-based primary MRP4 sequence determined lacks several crucial pieces of information including: (1) a classic Walker A motif which is a signature of the ATP-binding domain found in ABC cassette transporter members; (2) more than 90% of the protein sequence; and (3) any functional marker. MRP3, MRP4 and MRP5 have been localized to a different chromosome than MRP1 and MRP2, indicating that they are not merely alternative splicing products (49).
ABC transporters are integral membrane proteins involved in ATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multi-drug resistance. Many ABC transporters are predicted to consist of two functional domains, a membrane-spanning domain and a cytoplasmic domain. The latter contain conserved nucleotide-binding motifs with the former containing substrate binding or recognition sites. Attempts to determine the structure of ABC transporters and of their separate domains have not yet been successful (57).
The ABC transporters of glutathione S-conjugates and related amphiphilic anions have been identified as MRP1 and MRP2. These 190-kDa membrane glycoproteins have been cloned. MRP1 and MRP2 have been shown to be unidirectional, ATP-driven, export pumps with an amino acid identity of 49% in humans. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes. MRP2, also known as canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, such as the hepatocyte canalicular membrane and kidney proximal tubule luminal membrane. Physiologically important substrates of both transporters include glutathione S-conjugates, such as leukotriene C4, as well as bilirubin glucuronides, 17xcex2-glucuronosyl estradiol and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 studied in inside-out oriented membrane vesicles is very different from MDR1 (Pgp). MRP1 and MRP2 have been called conjugate transporting ATPases, functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress (54).
A cDNA encoding another ATP-binding cassette transporter, MOAT-B, has been reportedly cloned and mapped (56). Comparison of the MOAT-B predicted protein with other transporters revealed that it is most closely related to MRP. cMOAT, and the yeast organic anion transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a approximately 200 amino acid NH2-terminal hydrophobic extension that is present in MRP and cMOAT and which is predicted to encode several transmembrane spanning segments. In addition, the MOAT-B tissue distribution is distinct from MRP and cMOAT. In contrast to MRP, which is widely expressed in tissues, including liver, and cMOAT, the expression of which is largely restricted to liver, the MOAT-B transcript is widely expressed, with particularly high levels in prostate, but is barely detectable in liver. (6).
The sequence and structural similarity between eukaryotic and prokaryotic ABC transporters is striking. The sequence similarity extends beyond the conserved components (the nucleotide-binding sequence motif, i.e. Walker motifs, and the ABC signature sequence) and includes several hundred amino acids on either side of the Walker motifs. Functionally, this suggests conservation between the coupling of ATP binding and ATP hydrolysis, with both processes necessary to facilitate substrate transport. The functional conservation is observed from prokaryotic to eukaryotes even though the substrates may be markedly different. Further evidence that the ABC transporters can be grouped together is found in an analysis of their predicted secondary structures which shows these molecules possess remarkably similar secondary structures across the entire phylogenetic spectrum of ABC transporters.
The recently completed sequence of the yeast genome revealed that over 29 proteins belong to the ABC transporter family (59). Despite the sequence and structural similarity a diverse array of substrates and functions are attributed to the ABC transporters. For example, in S. cerevisiae, the STE6 protein is necessary for the secretion of essential mating protein, xcex1-factor; in D. melanogaster, the white and brown gene products may transfer pigment proteins: in mammals, MRP1 transports some cancer therapeutic agents and glutathione conjugates; and CFTR serves as a ion channel. Thus, it is clear that simply being a member of the large ABC transport family does not in any way define the type of substrate transported nor does a predicted secondary structure. Further, many of the substrates transported are structurally diverse and do not share clearly definable molecular signatures.
Antiviral therapies used to treat HIV and other DNA virus infections include acyclic nucleoside phosphonates. These represent a new class of nucleotide analogs that exhibit potent and selective activity against a variety of both DNA and RNA viruses, including the human immunodeficiency virus and hepatitis B virus.
The acyclic nucleoside phosphonate PMEA (9-(2phosphonylmethoxyethyl)adenine) is a broad-spectrum agent that exhibits potent antiviral activity against various DNA viruses and retroviruses, including HIV (1-14). PMEA and its lipophilic prodrug bispom-PMEA (15) have entered phase I clinical trials as treatment for HIV infections (6.17). PMEA acts as a stable monophosphate analog of AMP and dAMP, and its antiviral activity is thought to require activation to the diphosphate derivative PMEApp, which then acts to inhibit viral DNA polymerases with relative sparing of cellular DNA replication (8, 11, 18). However, the exact mode of metabolism and action of PMEA and related acyclic phosphonate analogs remains unclear. Studies have suggested that PMEA enters cells via endocytosis and is further metabolized to PMEAp and PMEApp by cellular enzymes (19). One report suggested that the anabolism of PMEA may involve direct conversion to PMEApp via PRPP synthetase, although direct evidence for this mechanism in intact cells has not been obtained (11).
Sequential intracellular phosphorylation of the nucleoside and phosphonate analogs is essential for the bioactivation of these compounds (6-8). The phosphorylated metabolites function as anti-HIV drugs by inhibiting the reverse transcriptase (RT) enzyme of HIV. It has been well documented that achieving optimal intracellular concentrations of the phosphorylated biotransformation products is important for exerting the anti-viral effect (9, 10).
For a clinically efficacious treatment of HIV infection, tong-term use of the nucleoside RT inhibitors is a very common practice. The development of drug resistance to these compounds is well documented in the literature. Several reasons for the development of drug resistance have been observed: (i) These nucleoside analogs and the acyclic nucleoside phosphonates select for various mutations in the RT that confers varying degrees of viral resistance to the different analogs (11, 12). (ii) A decrease in the enzymes involved in the biotransformation of the drug to the active metabolite leads to cellular resistance (13). (iii) A change in the transport of the drug, either decreased uptake or increased flux such that optimal intracellular levels are not attained could lead to cellular resistance (48).
Because PMEA is a nucleotide analog which enters cells by a nonspecific process of endocytosis, decreased cellular influx is unlikely as a mode of cellular resistance. Nevertheless, studies have revealed that resistance to PMEA and similar compounds (e.g. AZT) appears secondary to decreased intracellular accumulation, thus limiting the therapeutic effectiveness of these compounds. Moreover, different T-cell lines do not achieve comparable intracellular concentrations of these compounds despite comparable extracellular concentrations.
These findings are consistent with the suggestion that intracellular accumulation may negatively impact pharmacologic antiviral therapeutic efficacy and imply enhanced transport of these compounds out of the cell as an important unrecognized mechanism in therapeutic failure. Accordingly, the identification of inhibitors and substrates associated with drug efflux and the development of strategies for controlling this activity, would therefore be expected to have substantial therapeutic impact, and it is toward the fulfillment of this and other like objectives that the present invention is directed.
In its broadest aspect, the present invention relates to a protein hereinafter referred to and exemplified by multi-drug resistance protein 4 (MRP4), and extends to nucleic acid molecules encoding it, cells that express it, and to its variants, including conserved variants, antagonists including antibodies, analogs, and mimics, including small molecules.
The invention further relates to finding respecting the role of MRP4 in drug efflux particularly in humans, and to the diagnostic and therapeutic applications of this activity, such as the ability to enhance and extend drug and like therapies where efficacy is dependent upon the development and maintenance of consistent, high intracellular levels of the therapeutic agent. In such event, the invention extends to, e.g. methods for administering anti-cancer or anti-HIV therapeutic agents, which methods include modulating the expression of MRP4 and/or administering antagonists or other agents that suppress the drug efflux activity of MRP4.
Further, the invention extends to the novel human T cell line. termed CEMr-1 has been generated which expresses a multi-drug-resistant phenotype to a variety of unrelated clinically active anti-HIV nucleoside agents such as zidovudine (AZT), lamivudine (3TC), and the acyclic phosphonate analogs such as PMEA and PMPA. (48). This multi-drug-resistant phenotype is associated with an ATP-dependent efflux of the mono phosphorylated congeners from these cells. These results suggested the involvement of an efflux pump similar to those described for many cancer drugs. However, biochemical characterization of the resistant cells revealed that the known P-glycoprotein pump was not responsible for drug efflux in CEMr-1 cells
As stated above, the present invention further extends to and provides a method for modulating drug resistance by controlling the presence, activity and/or the expression of MRP4, its mimics, antagonists, analogs, congeners, active fragments, conserved variants, and mixtures. The present invention represents the first example of a role of MRP4 in drug resistance. Moreover and as stated above, these results suggest that MRP4 functions as an organic anion transporter that is capable of effluxing nucleoside analogs and other drugs with anionic functionality. Indeed this represents the first mammalian pump described for nucleoside analogs with anionic functionality (i.e. all analogs with a purine and pyrimidine selection). It is expected that certain patients who either develop resistance to therapy without displaying viral resistance may develop cellular resistance by this mechanism. Alternatively, variation between individual""s MRP4 expression may determine therapeutic efficacy.
The present invention discloses that MRP4 functions as a drug efflux protein. MRP4 antibodies are also disclosed by the instant invention, and their diagnostic and therapeutic use is also contemplated.
MRP4 may represent the first gene described in mammals that effluxes intracellular nucleotides, thus representing an important molecule regulating nucleotide balance within cells, with therapeutic relevance.
It is another object of the present invention to provide an antibody capable of specifically binding to the provided protein without substantially cross-reacting with non MRP4 proteins or homologs thereof under conditions permissive to antibody binding.
Also an object of the present invention is to provide a diagnostic kit for identifying individuals resistant to anti-retroviral agent therapy comprising the provided probe or antibody.
It is also an object of the present invention to provide a kit for identifying a compound which is refractive to MRP4 efflux comprising the provided probe.
It is an object of the present invention to provide a nucleic acid probe capable of specifically hybridizing with the provided nucleic acid.
Additionally, it is an object of the present invention to provide a method of identifying the provided MRP4 protein in a sample.
It is a further object of the present invention to provide a method for identifying a nucleic acid in a sample which encodes MRP4 protein.
Still a further object of the present invention is to provide a method for identifying a compound that modulates expression of MRP4.
Further still, it is an object of the present invention to provide a method for identifying a compound capable of modulating MRP4 protein activity.
Also an object of the present invention is to provide a method of modulating MRP4 protein activity in a sample, comprising contacting the sample with the modulator compound identified by the provided method.
Yet another object of the present invention is to provide a pharmaceutical composition which comprises the identified modulator compound and a pharmaceutically acceptable carrier.
Still a further object of the present invention is to provide a method for treating a condition in a subject which comprises administering to the subject an amount of the provided pharmaceutical composition, effective to treat the condition in the subject.
A further still object of the present invention is to provide a method for identifying subjects at risk for resistance to anti-microbial agent therapy.
Yet another object of the present invention is to provide a method for identifying an anti-microbial agent which is refractive to MRP4 efflux activity.
It is still further object of the present invention to provide a transgenic non-human animal whose somatic and germ cells contain and express a gene encoding MRP4 protein, the gene having been introduced into the animal or an ancestor of the animal at an embryonic stage and wherein the gene may be operably linked to an inducible promoter element.
Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description which proceeds with reference to the illustrative drawings.